, Volume 94, Issue 10, pp 847–852

Sexual contact influences orientation to plant attractant in Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)


    • Chemicals Affecting Insect Behavior Laboratory, Plant Sciences Institute, Agricultural Research ServiceUnited States Department of Agriculture
Short Communication

DOI: 10.1007/s00114-007-0261-z

Cite this article as:
Dickens, J.C. Naturwissenschaften (2007) 94: 847. doi:10.1007/s00114-007-0261-z


Chemical signals emitted by insects and their hosts are important for sexual communication and host selection. Plant volatiles facilitate the location of suitable hosts for feeding and oviposition, and may moderate responses to sex and aggregation pheromones. While mating has been shown to moderate behavioral responses to pheromones in a number of insects, little is known about the effects of mating on behavioral responses of insects to plant attractants, and even less is known about the mechanisms involved. In this study, mating was shown to decrease behavioral responses of the Colorado potato beetle to a host kairomone within 24 h, and attraction to the kairomone recovers only after 72 h. This decrease in responsiveness also occurs when only contact with the opposite sex is allowed; the effect is not observed with contact among individuals of the same sex. Peripheral olfactory responses to a component of the kairomone correlate with the observed behavioral responses and suggest involvement of antennal receptors in the behavioral change.


KairomoneBehavioral changeColorado potato beetleOlfactionLeptinotarsa decemlineataMatingSexual contact


Chemical signals play a major role in the orientation of insects to mates and suitable hosts. Sex pheromones such as those produced by female moths attract males for mating; aggregation pheromones emitted by many coleopterous species attract both sexes for both feeding and mating (Howse et al. 1998). Host volatiles released by plants may be attractive alone (Städler 1992), or may enhance or inhibit responses of insects to their sex or aggregation pheromones (Landolt and Phillips 1997).

Behavior of insects in response to chemical signals may be affected by a number of factors including mating status. For example, male moths do not respond to the female-produced sex pheromone after mating (Gadenne et al. 2001). Conversely, attraction of female moths to host plant odors increases after mating (Landolt 1989; Yang et al. 2005). Inhibition of response of male moths to sex pheromone involves decreased sensitivity of interneurons in the antennal lobe and is dependent on juvenile hormone (Anton and Gadenne 1999); sensitivity of antennal receptors for the pheromone remains unchanged after mating (Gadenne et al. 2001). Attraction of female moths to host plants after mating may also be controlled within the central nervous system as peripheral responses to components of an attractant kairomone are unchanged (Rajapaske et al. 2006). A different paradigm exists in Diptera where virgin females choose male-produced pheromone over host fruit odor; after mating females prefer host fruit odor (Jang 1995). In Coleoptera, responses of male khapra beetles to a female pheromone decreases after mating (Adeesan et al. 1969). To my knowledge, little is known about the effects of mating on behavioral or electrophysiological responses of beetles to host plant attractant kairomones.

Colorado potato beetles Leptinotarsa decemlineata (Say) are attracted to volatiles emanating from potato plants (McIndoo 1926). More recently, a number of host volatile blends were identified from potato, which were attractive to L. decemlineata adults (Dickens 1999, 2000). A three-component blend comprised of (Z)-3-hexenyl acetate, (±)-linalool, and methyl salicylate was attractive to both adults and immature stages (Dickens 2000, 2002). The activity of this three-component kairomone was verified in field tests and its potential usefulness in control strategies for pestiferous populations was demonstrated (Martel et al. 2005).

In this study, the effects of mating and sexual contact on behavioral responses of L. decemlineata are demonstrated to the aforementioned three-component kairomone. Electrophysiological recordings were made from antennal receptors in response to one of the components of the kairomone to examine the possibility that mating might have an effect on the peripheral receptors.

Materials and methods


Adult L. decemlineata were obtained from a laboratory colony, which was annually infused with feral insects. The insects were maintained on potato, Solanum tuberosum cultivar Kennebeck, for both larval and adult stages. Upon emergence, adults were sexed; individual insects were kept in translucent, but not transparent plastic containers with fresh potato foliage and water. Insects were held at 25°C and a photoperiod of 14 h L/10 h D in an incubator until use 6 to 12 days post emergence. Before testing, Colorado potato beetles were starved for 2 to 3 h. Experimental insects were used only once.

Odorous stimuli

Components of the plant attractant (Dickens 2000) were: (Z)-3-hexenyl acetate 98% purity from Sigma Chemical, St. Louis, MO, USA; (±)-linalool 97% purity, and methyl salicylate 99% purity both from Aldrich Chemical, St. Louis, MO, USA.

Behavioral studies

Orientation to the attractant blend was tested in an open Y-track olfactometer modified after Visser and Piron (1998) and described in detail by Dickens (1999, 2000). In brief, volatiles emanating from the attractant blend on filter paper discs (2.5 cm diameter, Whatman™ #1 filter paper) in Ehrlenmeyer flasks were delivered to either side of the device. The attractant blend was comprised of 25 μl of neat chemicals (8.33 μl of (Z)-3-hexenyl acetate, (±)-linalool, and methyl salicylate) that were diluted in 1 ml of mineral oil (ca. 25 μg total chemicals/μl mineral oil) (Dickens 2000). A 25-μl aliquot of a 10× dilution of the three-component blend (ca. 62.5 μg total chemicals) was applied to a filter paper disk placed in the Ehrlenmeyer flask, while 25 μl of mineral oil served as the solvent control. Hydrocarbon free air that was humidified by passing through distilled water carried the odor molecules to either arm of the bioassay apparatus. Both the treatment and the control were replenished after 30 min of use in the bioassay apparatus. Airflow was regulated to 1 l/min by flowmeters. Experiments were conducted in a darkened room at 22°C in which the only source of light was that associated with the bioassay device. Air was constantly removed from the room by a fume hood and replenished simultaneously by the air conditioning system.

Experimental groups tested in laboratory bioassays were: 1) males and females before mating (0 h, Fig. 1a); males and females 2 h, 1 day, 2 days, 3 days, and 4 days after mating; 2) males and females before contact with an individual of the opposite sex (0 h, Fig. 1b); males and females 2 h, 1 day, 2 days, 3 days, and 4 days after contact with an individual of the opposite sex but mating disallowed; and 3) males and females before contact with an individual of the same sex (0 h, Fig. 1c); males and females 2 h, 1 day, 2 days, 3 days, and 4 days after contact with an individual of the same sex. For each treatment, two insects of the same or opposite sex were placed in a Petri dish (20 mm height × 90 mm diameter) for 1 h or until mating occurred. Once mating ensued, the insect pair was allowed to remain in copula for the duration. Where mating was disallowed, the pair was separated immediately upon contact with the opposite sex. For all bioassays, at least 20 males and 20 females were tested.
Fig. 1

Effect of mating (a), contact with the opposite sex (b), and contact with individuals of the same sex (c) on behavioral responses of Leptinotarsa decemlineata adults to a synthetic plant attractant in a dual-choice olfactometer. “0 h” indicates response of males and females before the experimental treatments. Asterisks indicate that response to the plant attractant (indicated by solid dark bars) differs from response to the solvent control (indicated by stippled bars) (*P < 0.05; **P < 0.01). See text for details.


Sensitivity of antennal receptors was measured using electroantennograms (EAGs) (Schneider 1957; Dickens et al. 2002). Antennae severed from adult L. decemlineata were placed between two capillary electrodes filled with 0.1 M NaCl. Silver wires placed within the capillaries carried the electrical signal to the recording equipment comprised of a minicomputer equipped with software for acquisition and analyses of electrical signals from insect preparations (Syntech, Hilversum, The Netherlands). Mean EAGs in millivolts (n = 18, nine males and nine females) measured sensitivity of three experimental groups to serial stimulus loads (0.005–5 μg) of methyl salicylate, a component of the plant attractant (Dickens 2000). The three experimental groups were: unmated males and females; males and females 1 day after mating; and males and females 3 days after mating. Methyl salicylate was diluted in nanograde hexane. Five-μl aliquots of the dilutions were placed on strips of Whatman #1 filter paper (7 × 18 mm) that were inserted into Pasteur pipettes. Molecules evaporating from the filter paper were delivered into a continuous stream of humidified air (1 m/s) by a 1-s puff of charcoal-filtered air (0.3 m/s). Interstimulus time intervals were 2–3 min. Mean responses to the solvent control both before and after each dilution series were subtracted from EAGs to the experimental odorant. EAGs to the solvent control for male or female beetles did not differ for the groups unmated, 1 day or 3 days after mating (P < 0.05, n = 9 for each sex, time period, and treatment, Duncan’s New Multiple Range Test, Duncan 1955).

Statistical analyses

Laboratory bioassays were assessed for significant differences by the hypothesis on binomial proportions based on the standard normal approximation (Brase and Brase 1983). For electroantennogram studies, a three-way analysis of variance with subsequent means comparisons using SAS Proc Mixed with the Satterwaite option was conducted to test for EAG sensitivity effects due to gender, time after mating, and concentration (SAS Institute Inc. 2005). Heterogeneous within treatment variability was accounted for by assigning each time after mating and concentration treatment for each gender into one of four variance groups most representative of its within treatment variability. This allowed an appropriately sized standard error to be used in association with each treatment mean when making treatment comparisons.


Behavioral assays

Before the experimental treatments, male and female L. decemlineata were significantly attracted to the kairomone vs the solvent control (Fig. 1, 0 h). Mating reduced attraction of both male and female L. decemlineata to the kairomonal attractant (Fig. 1; *P < 0.05; **P < 0.01 by testing the hypothesis that the binomial proportion is significantly different from P = 50% using the standard normal approximation; Brase and Brase 1983). While no effect was apparent 2 h after mating, within 24 h there was no difference between attraction to the plant attractant vs the solvent control (Fig. 1a). This lack of attraction was maintained a second day before resumption of attraction 3 days after mating. Surprisingly, a similar effect was noted for both males and females that were allowed to contact individuals of the opposite sex but were not allowed to mate (Fig. 1b). Contact with individuals of the same sex produced no decrease in responses of either sex to the plant attractant (Fig. 1c).

Electrophysiological responses

Analysis of variance revealed two significant effects for stimulus load and time post mating (Table 1). EAGs increased significantly with stimulus load after reaching threshold at 0.5 μg without regard to treatment group (Fig. 2a, P = 0.0001, n = 18, SAS Proc Mixed with Satterwaite option, SAS Institute Inc. 2005). Regardless of gender or concentration, EAGs were statistically smaller for individuals 24 h post mating than for unmated individuals and those 3 days post mating, (Fig. 2b, P = 0.0102, n = 18, SAS Proc Mixed with Satterwaite option, SAS Institute Inc. 2005).
Table 1

Three-way analysis of variance (ANOVA) of factors affecting electroantennogram responses of Leptinotarsa decemlineata before mating, 24 h after mating, and 72 h following mating











Time post mating





Gender × time post mating





Stimulus load





Gender × stimulus load





Time post mating × stimulus load





Gender × time post mating × stimulus load





Asterisks indicate statistically significant effects.
Fig. 2

a Stimulus load effect on electroantennograms in millivolts of Leptinotarsa decemlineata in response to serial dilutions of methyl salicylate, a component of the synthetic plant attractant. Stimulus loads with different letters are significantly different. b Time-post-mating effect on electroantennograms in millivolts of L. decemlineata, which were unmated, 1 day after mating and 3 days after mating. Bars with different letters are significantly different. Vertical bars represent standard errors for a and b.


Mating results in lack of attraction of the L. decemlineata to the kairomonal attractant blend: (Z)-3-hexenyl acetate, (±)-linalool, and methyl salicylate. This reduction of attraction is not immediate, but rather only occurs 24 h after copulation; attraction to the kairomone then resumes 48 h later (3 days after copulation). The effect of mating on the coleopteran L. decemlineata response to the plant attractant differs from behavioral responses observed in Lepidoptera and Diptera. In Lepidoptera, mating enhances behavioral responses of female moths to host plant volatiles (Landolt 1989; Rojas 1999; Mechaber et al. 2002; Jang et al. 2005). In tephritid fruit flies, mating stimulates a behavioral switch in which virgin females responding preferentially to a male-produced pheromonal attractant over host fruit odor subsequently prefer host fruit odor over the pheromonal attractant (Jang 1995). This behavioral switch does not occur at times less than 24 h after mating. The accessory glands of males are involved in behavioral changes after mating in a tephritid fruit fly (Jang 1995), and recently a sex peptide isolated from accessory gland extracts was shown to be responsible for post-mating effects in Drosophila melanogaster (Chapman et al. 2003).

Significant decrease in antennal responses to a component of the kairomone, methyl salicylate, indicates that the modulation of the behavioral response may be occurring at the peripheral level. This differs from mechanisms proposed for Lepidoptera where the modulation of behavioral responses of male moths to female-emitted sex pheromone involves juvenile hormone-dependent decreases in sensitivity of interneurons in the antennal lobe (Anton and Gadenne 1999); responses of peripheral receptors as measured by EAGs stay the same (Gadenne et al. 2001). Similarly, EAGs of virgin and mated female moths do not differ, thus suggesting central nervous system involvement in the attraction of mated females to host plant attractants (Rajapakse et al. 2006).

If the behavioral change induced by mating or sexual contact in L. decemlineata is regulated at least partially by the antennal olfactory receptors, it may occur through changes in components of the peripheral olfactory machinery. In the malaria vector mosquito Anopheles gambiae, a candidate odor receptor is down-regulated after a blood meal (Fox et al. 2001); this down-regulation corresponded with reduction in olfactory responses in A. gambiae to human odors (Takken et al. 2001) as demonstrated earlier for sensory neural responses in the yellow fever mosquito Aedes aegypti (Davis 1984). A similar mechanism where mating induces down-regulation of odorant receptors for kairomonal molecules could account for changes in receptor responsiveness observed here for L. decemlineata.

A decreased response to plant attractants of both sexes of L. decemlineata after mating is adaptive. Adult male beetles produce an aggregation pheromone (Dickens et al. 2002) that is enhanced by host plant volatiles including the three-component kairomone used in this study (Dickens 2006). Once attracted to the host plant, mating ensues followed by egg deposition by fertilized females. Decreased levels of responsiveness to attractants might decrease movement of females from plants where males are located, thus ensuring mating. The likelihood that both sexes remain on the same plant is further enhanced as only contact is necessary for the change in responsiveness to the plant attractant. Alternatively, if down-regulation of odorant receptors is involved in the behavioral change, it may be economical as plant attractants are abundant. The restoration of sensitivity 72 h later may be functional as oviposition may have taken place and the likelihood of remating has increased.


I thank Dr. J. C. Davis for expert technical assistance; Dr. Bryan Vinyard, USDA, ARS, Biometrical Counseling Center, Beltsville, MD, for statistical analyses; Dr. T. H. Kuhar, Department of Entomology, Virginia Polytechnic Institute and State University, Painter, VA, for critical review of the manuscript; and Dr. A. Mattoo for support and encouragement. This research was funded in part by a grant from the USDA Potato Grants Program.

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